Anaplasma translocated substrate-1 (ATS-1) and sero-detection of anaplasma phagocytophilum

ABSTRACT

Disclosed is the use of isolated Ats-1 protein in  Anaplasma phagocytophilum  in the ELISA detection of  Anaplasma  pathogen. The recombinant expression of Ats-1 and its use as a kit for ELISA are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/399,639 filed Jul. 15, 2010, the contentof which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to the field of diagnosticassays for the detection of infectious agents in a mammal, includinghumans. Particular embodiments disclosed herein encompass Ats-1 proteinthat is useful in the sero-detection of Anaplasma phagocytophilum.

BACKGROUND OF THE INVENTION

Anaplasma phagocytophilum is a tick-borne pathogen responsible forgranulocytic anaplasmosis in humans (Bakken J. S., et al.: Humangranulocytic ehrlichiosis in the upper Midwest United States. A newspecies emerging? JAMA 272: 212-218, 1994). There has been a steady risein cases of Anaplasma infections, alone or through co-infection withother tick-borne pathogens (Varde S., et al.: Prevalence of tick-bornepathogens in Ixodes scapularis in a rural New Jersey County. Emerg.Infect. Dis. 4: 97-99, 1998). Left unchecked, Anaplasma infection can bea potentially fatal disease resulting from the targeting and replicationof the Anaplasma pathogen within human neutrophils (Bakken J. S. et al.:JAMA 272: 212-218, 1994). Anaplasma phagocytophilum infection thusemerges as a significant healthcare concern.

Detection of Anaplasma infection is crucial. Ideally, a diagnostic assayshould be capable of detecting Anaplasma infection at its early stages,when antibiotic treatment is most effective and beneficial. Traditionaldetection methods for Anaplasma infection includes: (i) microscopicidentification of morulae in granulocytes, (ii) PCR analysis using wholeblood, (iii) isolation of the Anaplasma bacterium from whole blood, and(iv) serological tests, particularly indirect immunofluorescence assay(IFA). Microscopic examination is tedious and prone to sampling error.PCR test is sensitive in detecting the tick-borne pathogen during theperiod of time when the pathogen is present in the blood of infectedpatients. IFA is most commonly used (Park, J., et al.: Detection ofantibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensisantigens in sera of Korean patients by western immunoblotting andindirect immunofluorescence assays. Clinical and Diagnostic LaboratoryImmunology 10(6): 1059-1064, 2003), but this test often gives falsepositive results. Such results can be attributed in part to the use ofwhole-cell antigens because such proteins may be shared with otherbacteria (Magnarelli, L. A., et al.: Use of recombinant antigens ofBorrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linkedimmunosorbent assays to detect antibodies in white-tailed deer. J.Wildlife Dis. 40(2): 249-258, 2004). When clinical symptoms aremanifested or high and stable antibody titers to Anaplasmaphagocytophilum are found in patient blood, it reaches a late infectionstage and bypasses the window of antibiotic treatment.

So far, there are only a few surface proteins on Anaplasma pathogen thatare used in diagnostic assay for immuno-responses (i.e., IgG and IgMresponses). It is generally believed that outer membrane proteins inpathogens are target for eliciting an immuno-response because they maybe the first to be exposed to immune cells of a host. Regarding theAnaplasma phagocytophilum species, U.S. Pat. No. 6,964,855 discloses theuse of an outer membrane protein and its fragments in a detection assay.U.S. Pat. No. 7,304,139 discloses a major surface protein 5 (MSP5) andits use in a diagnostic test. The '139 patent discloses a few patient'sreactivity towards MSP5 and it lacks any data relating sensitivity andspecificity, let alone any IgG/IgM distinction. Zhi et al. disclosescloning and expression of an outer membrane protein of 44 kDa and itsuse in a Western immunoblot assay (J. Clinical Microbiology 36(6):1666-1673, 1998). Both MSP5 and p44 are outer membrane proteins inAnaplasma phagocytophilum.

The host immune response to tick-borne pathogen infection is frequentlyvigorous, and it is typically an easy task for investigators to identifymany antigens (e.g., outer membrane proteins) which have been targetedas part of the host antibody response to infection. However, most ofthese antigens often fail when used as biomarkers for diagnosticpurposes. Hence, it is well established that there is often nocorrelation between protein antigenicity, to the extent such a parametercan even be accurately predicted, and whether or not a given proteinmight serve as a useful diagnostic marker. It is the present inventors'contention that the proteins most beneficial as biomarkers forinfection, and useful for assay development, are those that manage toevade the host immune response; successful identification of thesespecific antigens is a largely unmet challenge in the diagnosticsindustry, despite urgent needs for such biomarkers.

There remains a continuing need in the discovery of a novel antigenpresent in Anaplasma phagocytophilum that is useful and can provide ahighly specific and sensitive test for sero-detection of this pathogen.

SUMMARY OF THE INVENTION

The present inventors unexpectedly discovered a diagnostic assayemploying Ats-1 as an antigen to provide an accurate and sensitivediagnostic assay to detect Anaplasma infection. The finding issurprisingly because Ats-1 is an intracellular protein in Anaplasmaphagotycophilium and is secreted by the bacteria during Anaplasmainfection to weaken the host mitochondria.

The present invention provides an isolated protein of Anaplasmaphagocytophilum that is useful in the detection of Anaplasmaphagocytophilum. The isolated protein has an amino acid sequence of SEQID NO: 2. The present invention provides recombinant Ats-1 protein andmethods of using the protein in the detection of recent or ongoinginfections with Anaplasma phagocytophilum, which is useful in thediagnosis of human granulocytic anaplasmosis. The recombinant Ats-1protein has an amino acid sequence of SEQ ID NO: 2.

In one aspect, the present invention provides a polypeptide having anamino acid sequence set forth in SEQ ID NO: 2.

In another aspect, the present invention provides a compositioncomprising the isolated Ats-1 protein and a support. Preferably, thesupport may be polyethylene, polypropylene and glass. Preferably, thesupport is a microtiter well.

In another aspect, the present invention provides an isolatedpolynucleotide with nucleotide sequence set forth in SEQ ID NO: 1.

In one aspect, the present invention provides a vector comprising theisolated polynucleotide of Ats-1. The vector may be pET. The vector mayfurther comprise a promoter of DNA transcription operably linked to theisolated polynucleotides of interest. The vector may further comprise apromoter of DNA transcription operably linked to the isolatedpolynucleotides of interest. The vector may be pET, pENTR, orpCR®8/GW/TOPO®. The promoter may be a lac promoter, trp promoter or tacpromoter.

In one aspect, the present invention provides a host cell comprising thevector. The host cell may be E. coli; which may include NovaBlue K12strain or BL21 (DE3).

In one aspect, the present invention provides a method of preparing arecombinant protein of Ats-1 having an amino acid sequence of SEQ ID NO:2. The method comprises the steps of (i) introducing the isolated Ats-1gene into a host cell; (ii) growing the host cell in a culture undersuitable conditions to permit production of said recombinant protein;and (iii) isolating the recombinant protein of Ats-1.

In one aspect, the present invention provides a method of detecting thepresence of an antibody against Anaplasma phagocytophilum in abiological sample of a mammal, comprising: (i) immobilizing an isolatedprotein of Ats-1 onto a surface, the amino acid sequence of Ats-1 is setforth in SEQ ID NO: 2; (ii) contacting the isolated protein with apatient's biological sample, under conditions that allow formation of anantibody-antigen complex between the immobilized protein (antigen) andan antibody against Anaplasma phagocytophilum; and (iii) detecting theformation of the antibody-antigen complex; the detected antibody-antigencomplex is indicative of the presence of said antibody against Anaplasmaphagocytophilum in the biological sample.

In one aspect, the present invention provides a method of diagnosing aninfection of Anaplasma phagocytophilum in a mammal, comprising the stepsof (i) obtaining a biological sample from a mammal suspected of havingan Anaplasma phagocytophilum infection; (ii) immobilizing an isolatedAts-1 protein onto a surface, wherein said isolated protein has an aminoacid sequence set forth in SEQ ID NO: 2; (iii) contacting said isolatedprotein with said biological sample, under conditions that allowformation of an antibody-antigen complex; and (iv) detecting saidantibody-antigen complex, wherein the presence of said detectedantibody-antigen complex is indicative of an infection of Anaplasmaphagocytophilum in said mammal.

In one aspect, the mammal is a human. In another aspect, ELISA testemploys an IgG or IgM assay. Preferably, the IgG ELISA has a sensitivityof at least 90%, and a specificity of at least 80%. Preferably, the IgMELISA has a sensitivity of at least 90%, and a specificity of at least75%.

In yet another aspect, the present invention provides an article ofmanufacture comprising a packaging material and a recombinant Ats-1protein. The article of manufacture may further comprise an instructionfor detecting the presence of antibody against Anaplasmaphagocytophilum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of Anaplasma translocatedsubstrate-1 (Ats-1) (NCBI Accession No. FJ210653) (SEQ ID NO:1).

FIG. 2 depicts the amino acid sequence of Anaplasma translocatedsubstrate 1 (Ats-1) (NCBI Accession No. ACN39579) (SEQ ID NO:2).

FIG. 3 depicts EK/LIC PCR amplification (from Anaplasma genomic DNA) ofthe cDNA encoding Ats-1 protein of Anaplasma phagocytophilum.

FIG. 4 depicts a pET-30 vector containing the Ats-1 gene.

FIG. 5 depicts a colony PCR of transformants in NovaBlue E. coli.

FIG. 6 depicts a colony PCR of transformants in BL21 (DE3) E. Coli

FIG. 7 depicts a Coomassie-stained SDS PAGE gel showing the recombinantexpression of Ats-1 protein.

FIG. 8 depicts a Coomassie-stained SDS PAGE gel showing the Ni-NTAcolumn purification of recombinant Ats-1 protein.

FIG. 9 depicts Western blot detection with monoclonal anti-His tagantibody of His-tag labeled recombinant Ats-1.

FIG. 10 is a schematic depiction of the Sandwich ELISA using recombinantAts-1 for IgG and IgM antibody detection.

FIG. 11 depicts an IgG ELISA data using recombinant Ats-1 of Anaplasmaphagocytophilum.

FIG. 12 depicts a ROC analysis for recombinant Ats-1 IgG ELISA.

FIG. 13 depicts the Positive Predictive Value (PPV) and NegativePredictive Value (NPV) of the IgG ELISA for Recombinant Ats-1 ofAnaplasma phagocytophilum.

FIG. 14 depicts an IgM ELISA data using recombinant Ats-1 of Anaplasmaphagocytophilum.

FIG. 15 depicts a ROC analysis for recombinant Ats-1 IgM ELISA.

FIG. 16 depicts the Positive Predictive Value (PPV) and NegativePredictive Value (NPV) of the IgM ELISA for Recombinant Ats-1 ofAnaplasma phagocytophilum.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be better understood from the followingdescription of preferred embodiments, taken in conjunction with theaccompanying drawings. It should be apparent to those skilled in the artthat the described embodiments of the present invention provided hereinare merely exemplary and illustrative and not limiting.

DEFINITIONS

Various terms used throughout this specification shall have thedefinitions set out herein.

As used herein, “Ats-1” (Anaplasma translocated substrate-1) refers to aprotein having an amino acid sequence as set forth in SEQ ID NO: 2 (NCBIAccession No. ACN39579). The protein is present in Anaplasmaphagocytophilum and is encoded by a nucleotide sequence (NCBI AccessionNo. FJ210653). The Ats-1 protein is shown by the present inventors tobind to antibodies that are present in Anaplasma patients' sera in anELISA assay.

As used herein, the term “ELISA” refers to “Enzyme-Linked ImmunoSorbentAssay” and is a biochemical technique used in detecting the presence ofantibody or antigen in a sample.

As used herein, the term “IFA” refers to immunofluorescence assay. “IFAsero-positive sera from a patient” refers to sera (obtained from apatient) that exhibit positive immunofluorescence staining towards cellsthat have been infected with Anaplasma phagocytophilum. “IFAsero-negative sera from a patient” refers to sera (obtained from apatient) that exhibit negligible immunofluorescence staining towardscells that have been infected with Anaplasma phagocytophilum.

As used herein, the terms “protein,” “polypeptide” or “peptide” are usedinterchangeably.

As used herein, the term “recombinant protein” refers to a protein thatis recombinantly expressed by a host cell via the use of a vector thathas been modified by the introduction of a heterologous nucleic acid.For purposes of the present invention, these proteins are intended toencompass some protein variations insofar as they retain the ability tobind to antibodies present in Anaplasma infected patients in an ELISAassay with comparable sensitivity and specificity. One of an ordinaryskill in the art would appreciate that the protein variations mayinclude (i) conservative substitutions, (ii) substitution, (iii)addition, and (iv) deletion of amino acids. It would be furtherappreciated that a protein variant, when having a sufficiently high %amino acid sequence identity (e.g., >95%) and a similar antibody bindingactivity as to the parent protein, is intended to be encompassed by thepresent invention.

As used herein, the term “detecting” is used in its broadest sense toinclude both qualitative and quantitative measurements. For example, oneof the detecting method as described in this application is used toidentify the presence of Ats-1 in a biological sample. However, themethod can also be used to quantify the amount of Ats-1 in a biologicalsample and the quantity can be used to compare the Ats-1 levels fromdifferent biological samples.

As used herein, the term “detectable antibody” refers to an antibodythat is capable of being detected either directly through a labelamplified by a detection means, or indirectly through, e.g., anotherantibody that is labeled. For direct labeling, the antibody is typicallyconjugated to a moiety that is detectable by means such as abiotinylated antibody. Detection means refers to a moiety or techniqueused to detect the presence of the detectable antibody in the ELISAherein and includes detection agents that amplify the immobilized labelsuch as label captured onto a microtiter plate. For example, thedetection means is a fluorimetric detection agent such as avidin orstreptavidin.

As used herein, the term “mammal” refers to any vertebrate of the classmammalia, having the body more or less covered with hair, nourishing theyoung with milk from the mammary glands, and, with the exception of theegg-laying monotremes, giving birth to live young. Preferably, themammal is human.

As used herein, the term “primer” refers to a nucleotide sequence whichcan be extended by template-directed polymerization. For the purpose ofthis application, the term “nucleotide sequence” is intended to includeDNA or modification thereof.

As used herein, the term “biological sample” may include but are notlimited to blood (e.g., whole blood, blood serum, etc), cerebrospinalfluid, synovial fluid, and the like from a mammal such as a human ordomestic animal. Extraction of nucleic acids from biological samples isknown to those of skill in the art.

As used herein, the term “ROC” refers to Receiver OperatingCharacteristics Analysis. ROC analysis is a standard statistical toolfor evaluation of clinical tests. ROC accesses the performance of thesystem in terms of “Sensitivity” and “1-Specificity” for each observedvalue of the discriminator variable assumed as decision threshold (i.e.,cutoff value to differentiate between two groups of response). ForELISA, the cutoff value can be shifted over a range of observed values(i.e., OD_(450/620)nm reading), and Sensitivity and 1-Specificity can beestablished for each of these values. The optimal pair of Sensitivityand Specificity is the point with the greatest distance in a Northwestdirection.

In our earlier filed patent applications, the present inventors cloned,expressed, purified, and used recombinant type IV secretion system(TIVSS) proteins such as virB10 and virB11 (rTIVSS virB10 and rTIVSSvirB11) and its protein fragments (Ser. No. 12/658,268 filed Feb. 9,2010, and Ser. No. 12/658,506 filed Feb. 9, 2010) and hemolysin (Ser.No. 12/658,537 filed Feb. 9, 2010) in the development of a diagnosticELISA test useful for detecting IgM/IgG antibody responses to Anaplasmaphagocytophilum. The discovered assays discriminate between Anaplasmaphagocytophilum IFA-positive and IFA-negative patient samples with highsensitivity (generally >70%) and specificity (generally >90%) values.The disclosure of these applications is hereby incorporated by referencein its entireties.

The present invention provides an isolated Ats-1 protein and itspreparation thereof. The isolated Ats-1, when assayed in an ELISA assay,reacts to IFA sero-positive sera, indicating the presence of anti-Ats-1antibody in patient sera infected with Anaplasma phagocytophilum. Thespecificity of this response is revealed by the fact that isolated Ats-1does not react to IFA sero-negative sera from a patient infected withAnaplasma phagocytophilum.

Biology of ATS-1

Anaplasma phagocytophilum, the causative agent of human granulocyticanaplasmosis, is known to infect human neutrophils and inhibitmitochondria-mediated apoptosis. Specific bacterial factors involved inthis process have remained largely unknown. In a recent study by Niu etal. (2010), a genomic DNA library of Anaplasma phagocytophilum wasscreened for effectors of the type IV secretion system by a bacterialtwo hybrid system. Using Anaplasma phagocytophilum VirD4 as bait, Niuidentified a putative effector, named Anaplasma translocated substrate 1(Ats-1). Using triple immunofluorescence labeling and Western blotanalysis of infected cells, including human neutrophils, these authorsdetermined that Ats-1 is abundantly expressed by Anaplasmaphagocytophilum, translocated across the inclusion membrane, localizedin the host cell mitochondria, and cleaved ectopically. Theseinvestigators showed that expressed Ats-1 targeted mitochondria in anN-terminal 17 residue-dependent manner, localized in matrix or at theinner membrane, and was cleaved as native protein, which requiredresidues 55-57. In vitro-translated Ats-1 was imported in a receptordependent manner into isolated mitochondria. Ats-1 inhibitedetoposide-induced cytochrome c release from mitochondria, PARP cleavage,and apoptosis in mammalian cells, as well as Bax-induced yeastapoptosis. Ats-1(55-57) had significantly reduced anti-apoptoticactivity. Bax redistribution was inhibited in both etoposide-induced andBax-induced apoptosis by Ats-1. Taken together, Ats-1 is believed torepresent a bacterial protein that traverses five membranes and preventsapoptosis at the mitochondria.

Ats-1 is a cytoplasmic protein in Anaplasma, and remains such throughoutthe early stages of infection as it is translocated through the type IVsecretion system pilus to its final destination within the hostmitochondria. As Ats-1 remains cytoplasmically-bound, it is predicted tobe a poor antigen, if at all, by a host. In other word, Ats-1 is notexpected to be seen by the host immune system during Anaplasmainfection. The present inventors made a surprising discovery that Ats-1is an optimal usefulness biomarker for early and late stage Anaplasmainfection.

Recombinant Polypeptide of Ats-1

The present invention specifically contemplates expression andpreparation of recombinant and synthetic polypeptides, characterized bybeing capable of binding to antibodies present in IFA positive patientsera. The recombinant Ats-1 has an amino acid sequence as set forth inSEQ ID NO: 2. In one embodiment, the present invention thus comprisesthe isolated nucleic acid having the nucleotide sequence set forth inFIG. 1 (SEQ ID NO: 1). For purposes of this application, it is intendedto encompass other nucleotide sequences that exhibit nucleotidedegeneracy but produce the same amino sequence of Ats-1. The recombinantproteins of Ats-1 expressed by the nucleic acids described hereinencompasses the protein set forth in FIG. 2 (SEQ ID NO: 2). Therecombinant Ats-1 protein described herein possesses the ability to bindto antibodies present in IFA positive sera (and not IFA negative sera).

It is understood that these recombinant polypeptides encompass variants.One type of variants includes modification of amino acids of recombinantpolypeptides; such as, for example, substitution, deletion, or additionof amino acids. The present invention is intended to encompass thepolypeptide variants of Ats-1 that retain the antibody binding abilitytowards IFA sero-positive sera and do not react to IFA sero-negativesera from Anaplasma infected patients. One of ordinary skill in the artwould recognize that conservative amino acid substitutions may includesimply substituting glutamic acid with aspartic acid; substitutingisoleucine with leucine; substituting glycine or valine, or anydivergent amino acid, with alanine, substituting arginine or lysine withhistidine, and substituting tyrosine and/or phenylalanine withtryptophan. In another embodiment, addition and deletion of single aminoacid may be employed. It is also appreciated by one of ordinary skill inthe art that a few amino acids can be included or deleted from each orboth ends, or from the interior of the polypeptide without significantlyaltering the peptide's ability to bind antibody (i.e., maintain highsensitivity and specificity (>90%), when tested in an ELISA assay.

Recombinant Expression of Ats-1 Polypeptides: Vectors and Hosts

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell.

A DNA sequence is “operatively linked” or “operably linked” to anexpression control sequence when the expression control sequencecontrols and regulates the transcription and translation of that DNAsequence. The term “operatively linked” includes having an appropriatestart signal (e.g., ATG) in front of the DNA sequence to be expressedand maintaining the correct reading frame to permit expression of theDNA sequence under the control of the expression control sequence andproduction of the desired product encoded by the DNA sequence. If a genethat one desires to insert into a recombinant DNA molecule does notcontain an appropriate start signal, such a start signal can be insertedupstream (5′) of and in reading frame with the gene. A “promotersequence” is a DNA regulatory region capable of binding RNA polymerasein a cell and initiating transcription of a downstream (3′ direction)coding sequence. For purposes of defining the present invention, thepromoter sequence is bounded at its 3′ terminus by the transcriptioninitiation site and extends upstream (5′ direction) to include theminimum number of bases or elements necessary to initiate transcriptionat levels detectable above background. Within the promoter sequence willbe found a transcription initiation site (conveniently defined forexample, by mapping with nuclease S1), as well as protein bindingdomains (consensus sequences) responsible for the binding of RNApolymerase.

In one embodiment, the present invention provides the expression of theDNA sequences disclosed herein. As is well known in the art, DNAsequences may be recombinantly expressed by operatively linking thesequences to an expression control sequence in an appropriate expressionvector; and expressing that linked vector via transformation in anappropriate unicellular host. Such operative linking of a DNA sequenceof this invention to an expression control sequence, of course,includes, if not already part of the DNA sequence, the provision of aninitiation codon, ATG, in the correct reading frame upstream of the DNAsequence. A wide variety of host/expression vector combinations may beemployed in expressing the DNA sequences of this invention. Usefulexpression vectors, for example, may consist of segments of chromosomal,non-chromosomal and Synthetic DNA sequences. Suitable vectors includepET, pENTR, and pCR®8/GW/TOPO® and the like. The promoter contains lacpromoter, trp promoter and tac promoter.

In one embodiment, a host cell contains the vector comprising thepolynucleotides of the present invention. Exemplary host cell includesE. coli. Various E. coli strains include, for example, NovaBlue strain,BL21 (DE3) or BL21 pLsS (DE3).

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. However, one skilled in the art will be able to selectthe proper vectors, expression control sequences, and hosts withoutundue experimentation to accomplish the desired expression withoutdeparting from the scope of this invention. For example, in selecting avector, the host must be considered because the vector must function init. The vector's copy number, the ability to control that copy number,and the expression of any other proteins encoded by the vector, such asantibiotic markers, will also be considered. In selecting an expressioncontrol sequence, a variety of factors will normally be considered.These include, for example, the relative strength of the system, itscontrollability, and its compatibility with the particular DNA sequenceor gene to be expressed, particularly as regards potential secondarystructures. Suitable unicellular hosts will be selected by considerationof, e.g., their compatibility with the chosen vector, their secretioncharacteristics, their ability to fold proteins correctly, and theirfermentation requirements, as well as the toxicity to the host of theproduct encoded by the DNA sequences to be expressed, and the ease ofpurification of the expression products. Considering these and otherfactors, a person skilled in the art will be able to construct a varietyof vector/expression control sequence/host combinations that willexpress the DNA sequences of this invention on fermentation or in largescale animal culture.

For recombinant expression of the various proteins used in thisapplication, genes encoding the various proteins of interest can beconveniently inserted into a cloning vector and the vector containingthe gene of interest is transfected or transformed into a suitable hostcell for protein expression. Various publicly available vectors may beused. For example, vectors may include a plasmid, cosmid, viralparticle, or phage. Examples of vectors included pET30, pENTR,pCR8/GW/TOPO® and the like. Vector components generally include, but arenot limited to, one or more of a signal sequence, an origin ofreplication, a marker gene, an enhancer element, a promoter, and atranscription termination sequence. Construction of suitable vectorscontaining one or more of these components as well as the gene ofinterest employs standard ligation techniques which are known to theskilled artisan.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

Examples of suitable selectable markers for mammalian cells includethose that enable the identification of cells competent to take up theantigen-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979). The trp1 geneprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)).

A number of promoters can be used in order to enhance the expression ofthe gene of interest. In one embodiment, a promoter can be employedwhich will direct expression of a polynucleotide of the presentinvention in E. coli. Other equivalent transcription promoters fromvarious sources are known to those of skill in the art. Exemplarypromoters include the β-lactamase and lactose promoter systems (Chang etal., Nature, 275:615 (1978)), alkaline phosphatase, a tryptophan (trp)promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980)), and thelike.

A promoter may be operably linked to the protein-encoding nucleic acidsequence to direct mRNA synthesis. Promoters recognized by a variety ofpotential host cells are well known. For example, promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the protein of interest.

Transcription of a DNA encoding the antigen by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, whichcan act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to the15-kDa coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Anaplasma phagocytophilum antigen.

The nucleic acid (e.g., genomic DNA) encoding recombinant Anaplasmaphagocytophilum antigen of the present invention may be inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. For example, a full-length Ats-1 gene may be inserted into areplicable vector for cloning and for expression of full-length Ats-1protein or fragments thereof. The appropriate nucleic acid sequence maybe inserted into the vector by a variety of procedures. In general, DNAis inserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art.

Host cells are transfected or transformed with expression or cloningvectors described herein for antigen production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis, Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, Ca₂PO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., or electroporation isgenerally used for prokaryotes. For mammalian cells without such cellwalls, the calcium phosphate precipitation method of Graham and van derEb, Virology, 52:456-457 (1978) can be employed. Transformations intoyeast are typically carried out according to the method of Van Solingenet al., J. Bact., 130:946 (1977). However, other methods for introducingDNA into cells, such as by nuclear microinjection, electroporation,bacterial protoplast fusion with intact cells, or polycations, e.g.,polybrene, polyornithine, may also be used. For various techniques fortransforming mammalian cells, See Keown et al., Methods in Enzymology,185:527-537 (1990). The particular selection of host/cloning vehiclecombination may be made by those of skill in the art after dueconsideration of the principles set forth without departing from thescope of this invention (See, e.g., Sambrook et al., Molecular Cloning,A Laboratory Manual 2^(nd) edition, 1989, Cold Spring Harbor Press, NY).

The Ats-1 polypeptide (antigen) may be recombinantly produced as afusion polypeptide with a heterologous polypeptide. The heterologouspolypeptide may serve as a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the antigen-encoding DNA that is insertedinto the vector. In mammalian cell expression, mammalian signalsequences may be used to direct secretion of the protein, such as signalsequences from secreted polypeptides of the same or related species, aswell as viral secretory leaders. An overview of expression ofrecombinant proteins is found in Methods of Enzymology v. 185, Goeddel,D. V. ed. Academic Press (1990).

Recombinant gene expression may be measured in a sample directly, forexample, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Recombinant gene expression, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of cells ortissue sections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencepolypeptide or against a synthetic peptide based on the DNA sequencesprovided herein or against exogenous sequence fused to Anaplasmaphagocytophilum DNA and encoding a specific antibody epitope.

After expression, recombinant antigen may be recovered from culturemedium or from host cell lysates. If membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g. Triton-X100) or by enzymatic cleavage. Cells employed in expression of Anaplasmaphagocytophilum antigen can be disrupted by various physical or chemicalmeans, such as freeze-thaw cycling, sonication, mechanical disruption,or cell lysing agents.

It may be desired to purify recombinant antigen from host cell proteins.The following procedures are exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; reverse phaseHPLC; chromatography on silica or on a cation-exchange resin such asDEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; metalchelating columns to bind epitope-tagged forms of the protein ofinterest. Various methods of protein purification may be employed andsuch methods are known in the art and described for example inDeutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular antigenproduced.

Other than recombinantly expressed, Ats-1 protein may be purifieddirectly from cultured Anaplasma phagocytophilum. One skilled in the artwould recognize culturing Anaplasma phagocytophilum using standardmethods. One skilled in the art can also recognize methods to obtainpurified Ats-1 protein from cultured Anaplasma phagocytophilum. To thatend, cultured Anaplasma phagocytophilum can be pelleted using standardcentrifugation (e.g., 10,000×g for 10 minutes). Pelleted cells can bedisrupted by physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents (e.g.,BugBuster® Master Mix reagent). Methods to purify Ats-1 protein fromcultured cells are known in the art. Exemplary procedures include:fractionation on an ion-exchange column; reverse phase high performanceliquid chromatography (HPLC); liquid chromatography-mass spectrometry(LC-MS); chromatography on silica or on a cation-exchange resin such asDEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;immunoaffinity chromatography; immunoprecipitation; and the like.Various methods of protein isolation are described in Deutscher, Methodsin Enzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982).

ELISA Assay

Detection of antibody binding in IFA sero-positive sera may beaccomplished by techniques known in the art, e.g., ELISA (enzyme-linkedimmunosorbent assay), western blots, and the like. In one embodiment,antibody binding is assessed by detecting a label on the primaryantibody. In another embodiment, the primary antibody is assessed bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labeled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention. For example, toselect specific epitopes of recombinant or synthetic polypeptide, onemay assay antibody binding in an ELISA assay wherein the polypeptides orits fragments containing such epitope.

As appreciated by one skilled in the art, an enzyme-linked immunosorbentassay (ELISA) may be employed to detect antibody binding in IFAsero-positive sera. In an initial step of an ELISA, an antigen isimmobilized onto a surface (for example by passive adsorption known ascoating). For purposes of this application, exemplary antigens includeAnaplasma phagocytophilum Ats-1 protein, succinate dehydrogenase andp44-8 outer membrane protein and the like. Recombinant or purified(i.e., isolated) full-length protein as well as fragments thereof may beused. Immobilization of antigen may be performed on any inert supportthat is useful in immunological assays. Examples of commonly usedsupports include small sheets, Sephadex and assay plates manufacturedfrom polyethylene, polypropylene or polystyrene. In a preferredembodiment the immobilized antigens are coated on a microtiter platethat allows analysis of several samples at one time. More preferably,the microtiter plate is a microtest 96-well ELISA plate, such as thosesold under the name Nunc Maxisorb or Immulon.

Antigen immobilization is often conducted in the presence of a buffer atan optimum time and temperature optimized by one skilled in the art.Suitable buffers should enhance immobilization without affecting theantigen binding properties. Sodium carbonate buffer (e.g., 50 mM, pH9.6) is a representative suitable buffer, but others such as Tris-HClbuffer (20 mM, pH 8.5), phosphate-buffered saline (PBS) (10 mM, pH7.2-7.4) are also used. Optimal coating buffer pH will be dependent onthe antigen(s) being immobilized. Optimal results may be obtained when abuffer with pH value 1-2 units higher than the isoelectric point (pI)value of the protein is used. Incubation time ranges from 1-8 hours,overnight or 24 hours. Incubation may be performed at temperaturesranging from 4-37° C. Preferably, immobilization takes place overnightat 4° C. Preferably, Ats-1 is immobilized onto a surface (e.g., amicrotiter plate) at 4° C. overnight (i.e., 16 hours). The plates may bestacked and coated long in advance of the assay itself, and then theassay can be carried out simultaneously on several samples in a manual,semi-automatic, or automatic fashion, such as by using robotics.

Blocking agents are used to eliminate non-specific binding sites inorder to prevent unwanted binding of non-specific antibody to the plate.Examples of appropriate blocking agents include detergents (for example,Tween-20, Tween-80, Triton-X 100, sodium dodecyl sulfate), gelatin,bovine serum albumin (BSA), egg albumin, casein, non-fat dried milk andthe like. Preferably, the blocking agent is casein. The blockingtreatment typically takes place under conditions of ambient temperaturesfor about 0.5-4 hours, preferably 1 to 2 hours.

After coating and blocking, the plate is washed with TBST (0.05%Tween-20) and the protein is dried on the plate using a vacuum drier in37° C. for about 1 to 4 hours, preferably 2-3 hours. The plates are thencovered with plate sealers, and put in a dark sealer pouch and sealedwith a humidity sponge also in it. This can be stored in 4° C. for a fewmonths, depending on the stability of the protein.

Sera from the control (IFA sero-negative) or IFA sero-positive patientsare added to the immobilized antigens in the plate. Biological sample(i.e., sera) may be diluted in buffer. For IgM detection, human sera maybe diluted with casein dilution buffer at 1:50 to 1:400 dilutions.Preferably, human sera are diluted at 1:100. For IgG detection, humansera may be diluted with casein dilution buffer at 1:50 to 1:400dilutions. Preferably, human sera are at 1:200.5% Casein in 1× PhosphateBuffered Saline (PBS) containing 0.05% TWEEN 20® detergent may be used.TWEEN 20® acts as a detergent to reduce non-specific binding.

The conditions for incubation of the biological sample and immobilizedantigen are selected to maximize sensitivity of the assay and tominimize dissociation. Preferably, the incubation is accomplished at aconstant temperature, ranging from about 0° C. to about 40° C.,preferably from about 22 to 25° C. to obtain a less variable, lowercoefficient of variant (CV) than at, for example, room temperature. Thetime for incubation depends primarily on the temperature, beinggenerally no greater than about 10 hours to avoid an insensitive assay.Preferably, the incubation time is from about 0.5 to 3 hours. Morepreferably, the incubation time is 1-2 hours. Optimal time of 1 hour atroom temperature is found to maximize binding to immobilized captureantigen.

Following incubation of the biological sample and immobilized antigen,unbound biological sample is separated from the immobilized antigen bywashing. The solution used for washing is generally a buffer (“washingbuffer”) with a pH determined using the considerations and buffersdescribed above for the incubation step, with a preferable pH range ofabout 6-9. Preferably, pH is 7. The washing may be done three or moretimes. The temperature of washing is generally from refrigerator tomoderate temperatures, with a constant temperature maintained during theassay period, typically from about 0-40° C., more preferably about 4-30°C. For example, the wash buffer can be placed in ice at 4° C. in areservoir before the washing, and a plate washer can be utilized forthis step.

Next, the immobilized capture antigen and biological sample arecontacted with a detectable antibody at a time and temperature optimizedby one skilled in the art. Detectable antibody may include a monoclonalantibody or a polyclonal antibody. These antibodies may be directly orindirectly conjugated to a label. Suitable labels include moieties thatmay be detected directly, such as fluorochrome, radioactive labels, andenzymes, that must be reacted or derivatized to be detected. Examples ofsuch labels include the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, horseradish peroxidase(HRP), alkaline phosphatase (AP), glucose oxidase (GO), and the like.Preferably, the detection antibody is a goat anti-human IgG polyclonalantibody that binds to human IgG and is directly conjugated to HRP.Incubation time ranges from 30 minutes to overnight, preferably about 60minutes. Incubation temperature ranges from about 20-40° C., preferablyabout 22-25° C., with the temperature and time for contacting the twobeing dependent on the detection means employed.

The conjugation of such labels to the antibody, including the enzymes,is a standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al. “Methods forthe Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

Because IgG may occasionally interfere in IgM detection assays, IgG inpatient sera may be removed prior to IgM ELISA. One of ordinary skill inthe art would appreciate various methods of IgG removal from biologicalsamples (e.g., human sera). For example, commercial reagents such asGullSORB™ (Meridian Bioscience, Inc., Cincinnati, Ohio) may be used. IgGmay also be removed using Aurum Serum Protein Mini Kit (Bio-Rad,Hercules, Calif.), sucrose gradient sedimentation and the like.Preferably, IgG is removed using GullSORB®. Ideally, an anti-human IgGantibody is used to neutralize the IgG in human sera. The method for IgGremoval can be conveniently optimized by one of ordinary skill in theart. For example, human sera can be incubated with anti-human IgGantibody prior to the IgM ELISA assay.

Diagnostic Kits Employing Recombinant Ats-1 Polypeptides

The present invention provides a kit for the diagnosis of Anaplasmainfection. In one embodiment, the kit is an ELISA kit containingrecombinant polypeptides described herein, detection reagents includingprimary or secondary antibodies, and other necessary reagents includingenzyme substrates and color reagents. Additional components that may bepresent within such kits include an instruction detailing the detectionprocedure for Anaplasma phagocytophilum, using the recombinantpolypeptides of the present invention. The diagnostic kit of the presentinvention further comprises a positive and negative serum control. Thediagnostic kit of the present invention can also be used in diagnosingother infectious diseases involving Anaplasma phagocytophilum such asHuman Granulocytic Anaplasmosis (HGA).

The following Examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL STUDIES Example 1 Ats-1 in Anaplasma phagocytophilum

Using a genomic DNA library of Anaplasma phagocytophilum and a bacterialtwo-hybrid system, Niu et al. has recently disclosed a functional rolefor Ats-1 (˜48 kd bacterial protein). Immunofluorescence study revealsthat Ats-1 can be translocated across multiple membranes intomitochondria of the host cells (e.g., neutrophils). Upon arrival inmitochondria, Ats-1 is shown to inhibit etoposide-induced cytochrome Crelease, PARP cleavage, and apoptosis. These authors proposed that Ats-1represents a bacterial protein that traverses through cellular membranesand prevents apoptosis of the host cells in their mitochondria.

The present inventors surprisingly discovered a novel and unrelated rolefor Ats-1. We presented herein that Ats-1 is a good biomarker fordetecting Anaplasma infection in human, and for diagnosing Anaplasmainfection. Evidence is presented herein to demonstrate thatrecombinantly expressed Ats-1, when immobilized on a surface (e.g., inan ELISA assay), represents a good detection biomarker for both IgG andIgM antibody responses to Anaplasma phagocytophilum infection.

Example 2 Cloning and Recombinant Expression of Ats-1 and Six (6)Cytoplasmic Proteins in Anaplasma phagocytophilum

(i) PCR Amplification and Ligation into Plasmid Vector

To determine if Ats-1 protein may contain an epitope for antibodyrecognition, we cloned and recombinantly expressed Ats-1 protein inAnaplasma phagocytophilum.

Our cloning strategy involves designing and preparing syntheticoligonucleotides (˜30 bp in length) and used them to amplify the cDNAthat encodes the Ats-1 protein. For comparison purposes, we successfullycloned six (6) additional cytoplasmic proteins: including hemolysin,outer membrane protein (p44), and type IV secretion system proteins(i.e., VirB9, VirB10, VirB11, and VirD4) in Anaplasma phagocytophilum.

To this end, genomic DNA of Anaplasma phagocytophilum (a generous giftfrom Dr. S. Dumler at Johns Hopkins University) was used as the templatefor each of the PCR reactions. Synthetic oligonucleotides correspondingto eleven (11) genes were used for the PCR amplification reactions (See,Table 2). Using these synthetic oligonucleotides to amplify cDNAs fromgenomic Anaplasma phagocytophilum DNA, we successfully amplified nine(9) of the genes; namely, p44, virB3, virB6, virB9, virB10, virB11,virD4, succinate dehydrogenase and hemolysin; but failed to amplify two(2) genes (namely, virB4 and virB8) (See, Table 2).

Of the nine (9) amplified genes (Table 2), three (3) genes (namely,virB3, virB6 and succinate dehydrogenase iron-sulfur subunit) failed toexpress any recombinant proteins. Accordingly, in our hands, we wereable to amplify six (6) genes (that are related to type IV secretionsystem) and recombinantly express the respective proteins (Table 2).

Table 1 summarizes the nucleotide sequence accession numbers of the six(6) genes used in our PCR amplification reaction. The IgG and IgMresponses of the recombinant proteins (prepared using the respective six(6) genes) when used in ELISA are also shown (Table 1).

FIG. 3 shows an agarose gel of the amplified Ats-1 gene prior toprocessing of the PCR reaction in preparation for ligation into pET30vector. The amplicon having an expected size (˜1.16 kb) is shown by thearrow in this figure. In preparation for ligation with the vector, thePCR amplification reactions were treated to remove any remainingnucleotides, primers, and reaction components. The resulting PCRproducts were then treated with T4 DNA polymerase and ligated into pET30using standard protocols (See, FIG. 4). Ligation of insert DNA(including, e.g., virB3, virB6, virB9, virB10, virB11, virD4, succinatedehydrogenase iron-sulfur, hemolysin and p44 proteins) was performed asdescribed below.

T4 Polymerase Treatment of PCR Products and Ligation into pET30 Vector

In order to ligate the cloned insert DNA with the plasmid vector, it isnecessary to create compatible ends between the amplicon and the chosenvector (e.g., pET30 Ek/LIC). We generated overhangs compatible with theEk/LIC cloning vector on the insert DNA by T4 DNA polymerase treatmentof the PCR amplicon. We ligated the treated amplicon into the expressionvector to form pET30/insert DNA.

FIG. 4 depicts the pET30 vector containing the inserted gene (e.g.,full-length virB3, virB6, virB9, virB10, virB11, virD4, succinatedehydrogenase iron-sulfur, hemolysin and p44). The nucleotide sequencesof virB3, virB6, virB9, virB10, virB11, virD4, succinate dehydrogenaseiron-sulfur, hemolysin and p44 are publicly available and theiraccession numbers are listed in Table 2.

Transformation of Recombinant Clones into NovaBlue E. coli

In these series of experiments, we transformed the ligated DNAs(annealing reaction) into host bacterial cells (NovaBlue E. coli). Theligated DNAs included virB3, virB6, virB9, virB10, virB11, virD4amplicons as well as succinate dehydrogenase iron-sulfur, hemolysin andp44 amplicons. We chose NovaBlue E. coli because this bacterial strainis optimized for producing a stable cell line containing a recombinantinsert (see, NovaBlue Ek/LIC manual). Transformation into NovaBluecompetent E. coli (Novagen) was performed using standard protocols.First, appropriate numbers of 20 μl aliquots of competent cells wereprepared from −80° C., and allowed to thaw on ice for several minutes,followed by the addition of 1 μl of the annealing reaction and gentlestirring. The mixture was further incubated on ice for an additional 5minutes, followed by heating the tubes for 30 seconds in a 42° C. waterbath. The tubes were immediately placed on ice for 2 minutes. SOC (SuperOptimal broth with Catabolite repression medium, containing 2% w/vbacto-tryptone, 0.5% w/v bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 20 mM glucose) (at room temperature) was added into the tubes,and the reactions were further incubated for 1 hour at 37° C. withshaking (250 rpm). Cells were plated onto LB agar plates (containingkanamycin) and incubated at 37° C. overnight.

Colony PCR of NovaBlue Transformants

To confirm the successful transformation of insert DNA (pET30/insertDNA) in E. coli cells, we selected several colonies of each transformantgrown on LB plates (with kanamycin), and performed colony PCR using thesame set of Ek/LIC primers as in the amplification of the genes from theAnaplasma genomic DNA. An aliquot of each PCR reaction was analyzedusing agarose gel electrophoresis.

As an example, FIG. 5 shows agarose gel electrophoresis analysis ofeight of Ats-1 transformants in NovaBlue E. coli. Amplicons of expectedsize (1,161 bp) were observed following analysis of the PCR reactions.NovaBlue E. coli colonies containing the pET30/insert DNA were furthercultured in LB-kanamycin broth (for the isolation of plasmids).

Plasmid Mini-Preps

In order to confirm the presence and sequence accuracy of the clonedinsert DNA in the pET30 vector, we performed sequence analysis on therecombinant plasmids. The sequence analysis also provides informationthat the insert was in-frame of the upstream His-tag sequence. First, weisolated plasmid DNA from the transformed E. coli. Wizard Plus SVMinipreps DNA Purification system (Promega) was used according to themanufacturer's recommended protocol. The concentration (1OD_(260/280)=0.5 mg/ml) and the relative purity (OD_(260/280)) of theisolated plasmid DNA preparations were determined by spectrophotometricanalysis.

Sequencing Analysis of Insert DNA

We next performed sequence analysis on the isolated plasmid DNA usingthe Applied BioSystems 3130 Genetic Analyzer DNA Sequencing instrument.All of the insert DNA were confirmed to be accurate by BLAST analysisand in-frame. BLAST (Basic Local Alignment Search Tool) analysis of thesequences confirmed a match between each of the nucleotide sequences andthe published sequences of the respective Anaplasma phagocytophilumgenes.

Transformation of BL21 (DE3) E. coli with Recombinant Plasmids

After confirmation of the obtained recombinant plasmids, we proceeded totransform them into BL21 (DE3) competent E. coli (Novagen).Transformation was carried out by removing the appropriate number of 20μA aliquots of competent cells from −80° C., allowing the tubes to thawon ice for several minutes, followed by the addition of 1 μA of theplasmid preparation to the cells with gentle stirring. The mixture wasincubated on ice for 5 minutes, followed by heating of the tubes forexactly 30 seconds in a 42° C. water bath. The tubes were immediatelyplaced on ice for 2 min. SOC (room temperature) was added, and thereactions were further incubated at 37° C. for 1 hour at 250 rpm. Cellswere then plated onto LB agar plated (containing kanamycin) andincubated at 37° C. overnight.

Colony PCR of BL21 (DE3) Transformants

To confirm the successful transformation of recombinant pET30/insert DNAin BL21 (DE3) E. coli cells, we selected several colonies of eachtransformant grown on LB plates (with kanamycin), and performed colonyPCR using forward and reverse vector-specific primers. An aliquot ofeach PCR reaction was analyzed using agarose gel electrophoresis.

FIG. 6 shows agarose gel electrophoresis analysis of six (6) of Ats-1transformants in BL21 (DE3) E. coli. Amplicons of expected size (˜1,161bp) were observed following analysis of the PCR reactions. Several BL21(DE3) E. coli colonies containing the pET30/insert DNA were thenprocessed for recombinant expression.

Isolation and Purification of Recombinant Ats-1

Isolation of the expressed recombinant Ats-1 was performed usingBugBuster Master Mix (Novagen) according to the manufacturer's protocol.After IPTG induction, bacterial cells were harvested from liquidcultures by centrifugation at 3,000 rpm for 15 minutes. Recombinantnon-TIVSS protein was isolated both from supernatant and cell pellets.Cell pellets were re-suspended in 5 ml of BugBuster Master Mix (Novagen)by gentle vortexing. The resulting cell suspensions were incubated on arotating mixer for 20 minutes at room temperature. The mixtures werecentrifuged at 4° C. for 20 minutes at 16,000×g to remove the insolublecellular debris. The supernatant was transferred to a fresh tube for SDSPAGE analysis (See, FIG. 7).

The supernatant was then processed to isolate the soluble fraction.Soluble fraction purification was carried out by using Ni-NTA Buffer kit(Novagen) according to the manufacturer's protocol. An aliquot of thepurified inclusion body fraction was analyzed on an SDS PAGE gel. (See,FIGS. 8 & 9).

In addition to Ats-1 we also confirmed the successful transformation ofrecombinant pET30/insert DNA for virB3, virB6, virB9, virB10, virB11,virD4, succinate dehydrogenase iron-sulfur, hemolysin and p44.

Expression of Various Recombinant Proteins

FIG. 13 depicts a flow chart depicting the steps for IPTG induction ofrecombinant TIVSS and non-TIVSS proteins in BL21 E. coli. For expressionof various recombinant TIVSS (rTIVSS) proteins (for example, virB3,virB6, virB9, virB10, virB11, and virD4) and non-TIVSS proteins (forexample, succinate dehydrogenase iron-sulfur submit and p44), BL21 (DE3)E. coli were transformed with the pET30-rTIVSS plasmid DNA containingthe respective genes.

The expression was induced with IPTG as follows: 3 ml of LB brothcultures with kanamycin (30 μg/ml final concentration) were inoculatedwith BL21 transformed with pET30-rTIVSS plasmid. Cultures were grown tomid-log phase (OD₆₀₀=0.5) at 37° C. with shaking at 250 rpm. When thecultures reached mid-log, the entire 3 ml was added to 100 ml LB brothwith kanamycin (30 μg/ml final concentration) and allowed to grow tomid-late log phase (OD₆₀₀=0.5-1). When the cultures reached mid-late logstage, they were split into two separate 50 ml batches in 250 ml flasks.To one flask, 500 μl of IPTG was added (final concentration of 1 mM),this flask being the induced flask. No IPTG was added to the other flaskwhich served as a control for assessing induction—the uninduced flask.Growth of the IPTG and control cultures was allowed to proceed for 4-4.5hours at 37° C. with shaking (250 rpm). Cell pellets were then harvestedby centrifugation at 3,000 rpm for 15 minutes at 4° C., and subsequentlyprocessed with BugBuster Master Mix (Novagen) as described below.

Recombinant Expression of virB3, virB6, and Succinate DehydrogenaseIron-Sulfur Subunit Fail

After IPTG induction and BugBuster Master Mix treatment, equalconcentrations (˜3 μg) of a soluble cytoplasmic and insoluble (inclusionbody) fraction from IPTG-treated (induced) cells and control cells wereanalyzed on SDS-PAGE. SDS-gels were stained using Coomassie-blue.Induction of recombinant protein expression was considered to besuccessful when there was a marked increase (observed on SDS-PAGEprotein gels) in the target protein expression in the IPTG-treatedsample, as compared to that of the control cells (i.e., no IPTG).

FIG. 7 shows that IPTG induction of Ats-1 (soluble and inclusion body)before and after IPTG induction. Note that Ats-1 shows marked inductionrelative to the control (uninduced), and the induced Ats-1 ispredominantly sequestered within the soluble fraction (see arrow).

Table 2 summarizes the results of recombinant expression of TIVSS andnon-TIVSS proteins. Using our expression protocol, we found that virB3,virB6 and succinate dehydrogenase fail to express any recombinantprotein.

Altogether, our results show that virB4 and virB8 genes could not beamplified under these experimental conditions. Unexpectedly, virB3 andvirB6 failed to recombinantly express their corresponding proteins. Wewere successful in recombinantly express only four (4) of the eight (8)TIVSS protein components (namely, virB9, virB10, virB11, and virD4) inAnaplasma phagocytophilum. In addition, we were only able torecombinantly express p44 outer membrane protein and hemolysin, but notsuccinate dehydrogenase iron-sulfur subunit (See, Table 2).

Example 3 IgG/IgM ELISA for Recombinantly Expressed Ats-1 Protein

We adopted IgG and IgM ELISA assays and evaluated the binding activityof the recombinant proteins towards IgG and IgM. The ELISA procedureinvolves: (i) coating 96-well micro-titer plates with the recombinantprotein at varying concentrations at 4° C. overnight; (ii) adding caseinin PBS to block non-specific binding; (iii) drying plates and keepingthem in 4° C. for long term storage; (iv) adding patients' sera to allowformation of antibody-antigen complex; (v) detecting theantibody-antigen complex. IFA sero-positive sera served as positivecontrols, and IFA sero-negative sera served as negative controls.Detection of antibody-antigen complex was performed with the use ofhorseradish peroxidase.

a) Patient Study: Ats-1 IgG ELISA

We conducted IgG ELISA tests for binding activity towards therecombinantly expressed Ats-1 protein. FIG. 10 shows a depiction of thatsandwich ELISA.

We examined recombinant Ats-1 in an IgG ELISA. Recombinant Ats-1 wasprepared using the cloning-expression method detailed above. Whentested, we observed a dose-dependent increase in the binding activity(as measured by OD_(450/620nm)) towards IgG sero-positive sera (FIG.11). The sensitivity of the IgG ELISA for recombinant Ats-1 was found tobe 100%. The specificity of the IgG ELISA was 80.6% (See, FIG. 11). Athreshold level of ≧70% is normally considered by industrial standard tobe meaningful and acceptable for accurate interpretation of ELISAsensitivity or specificity.

Because Ats-1 provides adequate IgG ELISA, we analyzed ROC (area underthe curve) using the raw IgG ELISA data with the MedCalc statisticalsoftware. FIG. 12 summarizes the performance analysis of the ROC curve.FIG. 13 shows the Positive Predictive Value (PPV) and the NegativePredictive Value (NPV) for the IgG ELISA using the recombinant Ats-1.

b) Patient Study: Ats-1 IgM ELISA

In this second series of studies, we examined recombinant Ats-1 in IgMELISA. FIG. 10 shows a depiction of that sandwich ELISA. RecombinantAts-1 protein exhibited a dose-dependent increase in binding towards IgMsero-positive serum (as measured by OD_(450/620nm)). IgM ELISA forrecombinant Ats-1 attained a 100% sensitivity (FIG. 14) and 75.9%specificity, both of which satisfies the threshold (≧70%) required byindustry.

The raw IgM ELISA data was analyzed with ROC curve determination usingMedCalc statistical software. Performance analysis of ROC curve is shownin FIG. 15. FIG. 16 shows the Positive Predictive Value (PPV) and theNegative Predictive Value (NPV) for the IgM ELISA using the recombinantAts-1.

Experimental Protocols

Induction and Purification of Ats-1:

-   -   1. Add a loop full of frozen stock of Ats-1 in BL21(DE3) cells        in 3 ml LB-Kan 30 culture and incubate at 37° C. shaking at 250        rpm until OD₆₀₀ is approximately 0.5. Add the entire 3 ml        culture to 100 ml LB-Kan medium in a 500 ml baffled flask.    -   2. Shake the culture in 37° C. incubator at 250 rpm until the        OD₆₀₀ is approximately 0.5-1.0    -   3. After the desired OD has been reached, take an aliquot of the        culture and label it “Uninduced”.    -   4. Induce the culture by adding 1 ml of 100 mM IPTG and return        to shake in 37° C. incubator at 250 rpm for 4 hours. After 4        hours, take an aliquot from the culture and label it “Induced”.        Analyze induced and uninduced samples on an SDS-PAGE gel to        observe the expression of the protein    -   5. Harvest cells from induced culture by centrifugation at        10,000×g for 10 minutes using a weighed centrifuge tube. Decant        the supernatant and allow the pellet to drain as much liquid as        possible.    -   6. Resuspend the cell pellet in room temperature BugBuster®        Master Mix reagent by pipetting or gentle vortex, using about        2.5 ml per 50 ml of culture.    -   7. Incubate the suspension on a shaking platform or rotating        mixer at a slow setting at room temperature for 20 minutes.    -   8. Centrifuge the cell debris at 16,000×g for 20 minutes at        4° C. to separate soluble and insoluble fractions.    -   9. Transfer the supernatant (soluble fraction) to a fresh,        sterile tube. Ats-1 is found in the soluble fraction.    -   10. Buffers in the Novagen Ni-NTA kit come as 4×. Dilute only        necessary amounts using distilled water    -   11. Add resin to the binding buffer (equal to 1 ml resin for        each 4 ml of soluble fraction)    -   12. Shake it for 5 min in 4° C. and let it settle for a few        minutes on ice    -   13. Take out the binding buffer from the top previously added        and add the soluble fraction. Let it shake in 4° C. for 1 hour    -   14. Pour in column, and slowly let flow through in tube on ice:        “Flow Through”    -   15. When finished collecting flow through, add 2×4 ml of wash        buffer and collect in tube on ice: “Wash Buffer1” and “Wash        Buffer2”    -   16. When finished collecting wash buffer, add 0.6 ml of elution        buffer 18 times and collect in 18×1.5 ml Eppendorf tubes:        “Elutions 1-18”    -   17. Save some of the leftover resin (put in PBS) to run on gel        along with the flow through, wash buffer, and elutions. Run gels        in double (one gel stain with coomassie and use the other for        western blot probed with a His-TAG antibody).    -   18. Combine the elutions that contain Ats-1. Buffer exchange it        in PBS, and read the concentration of the protein.

Anaplasma Ats-1 IgG ELISA

-   -   1. Antigen coating concentration 1.0 μg/ml in carbonate buffer        (pH 9.6) (100 μl per well). Coating overnight in 4° C.    -   2. Wash three time in PBST buffer (0.05% Tween-20)    -   3. Block with 200 μl blocker buffer (casein in PBS, Thermo Sci.        #37528). Incubate for 1 hour in room temperature    -   4. Wash three times with PBST buffer (0.05% Tween-20)    -   5. Add 100 μl 1:200 diluted human sera (dilution buffer: 1:20        casein buffer in PBST (0.05% Tween-20). Incubate for 1 hour in        room temperature    -   6. Wash four times with PBST buffer (0.05% Tween-20)    -   7. Add goat anti-human IgG antibody (1:15,000 diluted in casein        dilution buffer (1:20 casein buffer in PBST (0.5% Tween-20)).        Incubate for 1 hour in room temperature    -   8. Wash four times with PBST buffer (0.05% Tween-20)    -   9. Add 100 μA TBM substrate. Incubate in room temperature for 5        minutes 10 seconds in the dark.    -   10. Stop the reaction with 0.5M H₂SO₄    -   11. Read the result at OD_(450/620nm)

Anaplasma Ats-1 IgM ELISA

-   -   1. Antigen coating concentration 0.5 μg/ml in carbonate buffer        (pH 9.6) (100 μl per well). Coating overnight in 4° C.    -   2. Wash three time in PBST buffer (0.05% Tween-20)    -   3. Block with 200 μl blocker buffer (casein in PBS, Thermo Sci.        #37528). Incubate for 1 hour in room temperature    -   4. Wash three times with PBST buffer (0.05% Tween-20)    -   5. Dilute human sera in GullSorb™ (1:10) to prepare mixture 1.        Incubate in room temperature for 5 minutes. Dilute incubated        mixture 1 in sample dilution buffer (1:20 casein buffer in PBST        (0.5% Tween-20)). Therefore, the total dilution factor for human        sera is 1:100    -   6. Add 100 μl 1:100 diluted human sera to the plate. Incubate        for 1 hour in room temperature    -   7. Wash four times with PBST buffer (0.05% Tween-20)    -   8. Add goat anti-human IgM antibody (1:10,000 diluted in casein        dilution buffer (1:20 casein buffer in PBST (0.5% Tween-20)).        Incubate for 1 hour in room temperature    -   9. Wash four times with PBST buffer (0.05% Tween-20)    -   10. Add 100 μl TBM substrate. Incubate in room temperature for 5        minutes in the dark.    -   11. Stop the reaction with 0.5M H₂SO₄    -   12. Read the result at OD_(450/620nm)

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Various modifications andvariations of the described composition, method, and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Various modifications of the above-described modes for carrying out theinvention that are obvious to those skilled in the filed of molecularbiology, recombinant expression and related fields are intended to bewithin the scope of the following claims.

TABLE 1 Accession number: IgG ELISA IgM ELISA p44 YP_504769 (42 Samples)Not tested Sens: 42.9% Spec: 100% VirB9 YP_505897 Checkerboard - no (42Samples) conditions showed Sens: 61.9% good differentiation Spec: 100%VirB10 YP_505896 (42 Samples) (42 Samples) Sens: 81.0% Sens: 71.4% Spec:71.4% Spec: 90.5% VirB11 YP_505895 (42 Samples) Not tested Sens: 71.4%Spec: 76.2% VirD4 YP_505894 (42 Samples) Not tested Sens: 81.0% Spec:42.9% Hemolysin YP_504658 (42 Samples) (42 Samples) Sens: 81.0% Sens:76.2% Spec: 57.1% Spec: 90.5%

TABLE 2Oligonucleotide Sequences Used in Gene Amplification for Anaplasma phagocytophilumEncoding TIVSS and Non-TIVSS Protein Components Recombinant TIVSS & GeneRecombi- Non-TIVSS NCBI Amplifi- nant Protein Accession #Oligonucleotides cation Expression virB3 YP_504978Fwd: 5′-gacgacgacaagatgtctggtagtgtaaaagcg-3′ Yes No (Seq. ID No. 1)Rev: 5′-gaggagaagcccggtctacatcacatcataggaattag-3′ (Seq. ID No. 2) virB4YP_504979 Fwd: 5′-gacgacgacaagatgttaaagctaggttggtcttcg-3′ No No(Seq. ID No. 3) Rev: 5′-gaggagaagcccggtctatgcatttttcacccttg-3′(Seq. ID No. 4) virB6 YP_504980Fwd: 5′-gacgacgacaagatgcatagggtagcaagggcattg-3′ Yes No (Seq. ID No. 5)Rev: 5′-gaggagaagcccggtctaactctgaccaccttttcc-3′ (Seq. ID No. 6) virB8YP_505898 Fwd: 5′-gacgacgacaagatggtattggatatgtttggtc-3′ No No(Seq. ID No. 7) Rev: 5;40 -gaggagaagcccggtttatagaaattcatcatc-3′(Seq. ID No. 8) virB9 YP_505897Fwd: 5′-gacgacgacaagatgatgaatttctataaaaatttttatg-3′ Yes Yes(Seq. ID No. 9) Rev: 5′-gaggagaagcccggtctaactaagagcctgattc-3′(Seq. ID No. 10) virB10 YP_505896Fwd: 5′-gacgacgacaagatggctgacgaaataaggggttc-3′ Yes Yes (Seq. ID No. 11)Rev: 5′-gaggagaagcccggtctacctcaccgcatcacg-3′ (Seq. ID No. 12) virB11YP_505895 Fwd: 5′-gacgacgacaagatgactgggggtggtgcagctttag-3′ Yes Yes(Seq. ID No. 13) Rev: 5′-gaggagaagcccggtttacttattaccctctgaacacttagtgaac-3′ (Seq. ID No. 14) virD4YP_505894 Fwd: 5′-gacgacgacaagatgcatagttccaatcatatacg-3′ Yes Yes(Seq. ID No. 15) Rev: 5′-gaggagaagcccggtctactttagtcttccgttac-3′(Seq. ID No. 16) Succinate YP_504786Fwd: 5′-gacgacgacaagatggtgcagttttctttgcc-3′ Yes No Dehydrogenase,(Seq. ID No. 17) iron-sulfurRev: 5′-gaggagaagcccggtctagagctccaatccttttatc-3′ subunit(Seq. ID No. 18) Hemolysin YP_504658Fwd: 5′-gacgacgacaagatgggtgctggagtttttgaag-3′ Yes Yes (SEQ ID No. 1)Rev: 5′-gaggagaagcccggttcagcaagcagtattcctattcac-3′ (SEQ ID No. 2) p44-8YP_504769 Fwd: 5′-gacgacgacaagatgctaaggctcatggtgatgg-3′ Yes Yes Outer(Seq. ID No. 19) MembraneRev: 5′-gaggagaagcccggttcaaaaacgtattgtgcgacg-3′ Protein (Seq. ID No. 20)

1. A method of detecting the presence of an antibody against Anaplasmaphagocytophilum in a biological sample of a mammal, comprising the stepsof: (i) immobilizing an isolated Anaplasma translocated substrate-1(Ats-1) protein onto a surface; (ii) contacting said immobilized Ats-1protein with a biological sample from a patient suspected of containingan antibody against Anaplasma phagocytophilum, under conditions thatallow formation of an antibody-antigen complex; and (iii) detecting theformation of said antibody-antigen complex, wherein said detectedantibody-antigen complex is indicative of the presence of said antibodyagainst Anaplasma phagocytophilum in said biological sample.
 2. Themethod of claim 1, wherein said mammal is a human.
 3. The method ofclaim 1, wherein said isolated Ats-1 protein is a recombinant protein.4. The method of claim 1, wherein said support is selected from thegroup consisting of polyethylene, polypropylene and glass.
 5. The methodof claim 1, wherein said support is a microtiter well.
 6. The method ofclaim 1, wherein said biological sample is whole blood.
 7. The method ofclaim 1, wherein said antibody is an IgG.
 8. The method of claim 1,wherein said antibody is an IgM.
 9. The method of claim 1, wherein saidmethod is an ELISA.
 10. The method of claim 9, wherein said ELISA has asensitivity of at least 90%.
 11. The method of claim 9, wherein saidELISA has a specificity of at least 75%.
 12. A method of diagnosing aninfection of Anaplasma phagocytophilum in a mammal, comprising the stepsof: (i) obtaining a biological sample from a mammal suspected of havingan Anaplasma phagocytophilum infection; (ii) immobilizing an isolatedAnaplasma translocated substrate-1 (Ats-1) protein onto a surface; (iii)contacting said immobilized Ats-1 protein with said biological sample,under conditions that allow formation of an antibody-antigen complex;and (iv) detecting said antibody-antigen complex, wherein the presenceof said detected antibody-antigen complex is indicative of an infectionof Anaplasma phagocytophilum in said mammal.
 13. The method of claim 12,wherein the mammal is a human.
 14. The method of claim 12, wherein saidisolated Ats-1 protein is a recombinant protein.
 15. The method of claim12, wherein said support is selected from the group consisting ofpolyethylene, polypropylene and glass.
 16. The method of claim 12,wherein said support is a microtiter well.
 17. The method of claim 12,wherein said biological sample is whole blood.
 18. The method of claim12, wherein the antibody is IgG.
 19. The method of claim 12, wherein theantibody is an IgM.
 20. The method of claim 12, wherein said method isan ELISA.
 21. The method of claim 20, wherein said ELISA has asensitivity of at least 90%.
 22. The method of claim 20, wherein saidELISA has a specificity of at least 75%.
 23. An article of manufacturecomprising a packaging material and a recombinant Ats-1 protein.
 24. Thearticle of manufacture of claim 23, wherein said package materialfurther comprises an instruction for detecting the presence of anantibody against Anaplasma phagocytophilum.
 25. A composition comprisinga recombinant Ats-1 protein and a support, wherein said recombinantAts-1 protein has an amino acid sequence of SEQ ID NO:
 2. 26. Thecomposition of claim 25, wherein said recombinant Ats-1 protein isencoded by a polynucleotide having a nucleotide sequence of SEQ IDNO:
 1. 27. The composition of claim 25, wherein said support is selectedfrom the group consisting of polyethylene, polypropylene and glass. 28.The composition of claim 25, wherein said support is a microtiter well.29. A method of preparing a recombinant Anaplasma translocatedsubstrate-1 (Ats-1) protein, comprising the steps of: (i) introducing anisolated polynucleotide into a host cell, said isolated polynucleotidehaving a nucleotide sequence of SEQ ID NO: 1; (ii) growing said hostcell in a culture under suitable conditions to permit production of saidrecombinant Ats-1 protein; and (iii) isolating said recombinant Ats-1protein.
 30. The method of claim 29, wherein said recombinant Ats-1protein having an amino acid sequence of SEQ ID NO:
 2. 31. The method ofclaim 29, wherein said growing step further comprising the step ofadding Isopropyl-beta-D-thiogalactopyranoside (IPTG) to said culture.32. The method of claim 29, wherein said host cell is E. coli.
 33. Themethod of claim 32, wherein said E. coli is NovaBlue K12 strain, BL21(DE3) or BL21 pLyss (DE3).